Family GH-61 polypeptides

ABSTRACT

The present invention relates to use of an anti-staling GH-61 polypeptide for preparing an edible product.

CROSS REFERENCE

This application claims priority or the benefit under 35 U.S.C. 119 ofU.S. Provisional Application Nos. 60/491,131 and 60/417,733, filed onJul. 29, 2003 and Oct. 9, 2002, respectively, and Danish applicationnos. PA 2003 01096 and PA 2002 01459, filed on Jul. 22, 2003 and Oct. 1,2002, respectively, which are hereby incorporated by reference.

The present application contains information in the form of a sequencelisting, which is appended to the application and also submitted on adata carrier accompanying this application. The contents of the datacarrier are fully incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to the use of a GH 61 polypeptide forpreparing an edible product. The invention also relates to polypeptidesbelonging to the GH 61 family of polypeptides which improves theproperties of the edible product. The invention further relate topolynucleotides encoding said polypeptides, to nucleic acid constructscomprising such polynucleotides and to expression vectors andrecombinant host cells comprising such constructs. The invention alsorelates to processes for preparing said polypeptides and to compositionscomprising said polypeptides.

BACKGROUND OF THE INVENTION

Carbohydrates and glycol-conjugates are substrates for glycosyltransferases (GTs) and glycoside hydrolases (GHs). The structure ofglycoside hydrolases began to be solved starting from the 1980s. At thesame time, new GH proteins were discovered and their amino acid sequencedetermined. Two main observations emerged from the new data. 1) Theclassical E.C. nomenclature system for naming enzyme families was notprecise enough to classify the increasing number of enzymes that haddifferent structure yet performed the same enzymatic reaction. 2)Enzymes related by homology could have different enzymatic activity thusalso making the E.C. nomenclature system confusing for these relatedenzymes. A new family based nomenclature system was proposed by BernardHenrissat in 1991 based on the structure of the enzymes (Henrissat B., Aclassification of glycosyl hydrolases based on amino-acid sequencesimilarities. Biochem. J. 280:309–316(1991); Henrissat B., Bairoch A.New families in the classification of glycosyl hydrolases based onamino-acid sequence similarities. Biochem. J. 293:781–788(1993);Henrissat B., Bairoch A. Updating the sequence-based classification ofglycosyl hydrolases. Biochem. J. 316:695–696(1996) and Davies G.,Henrissat B. Structures and mechanisms of glycosyl hydrolases. Structure3:853–859(1995).). The classification of glycoside hydrolases infamilies based on amino acid sequence similarities was introducedbecause there is a direct relationship between sequence and foldingsimilarities, and such a classification is expected to:

-   -   (i) reflect the structural features of the enzymes, which cannot        be reflected by the substrate specificity alone,    -   (ii) help to reveal the evolutionary relationships between the        enzymes, and    -   (iii) provide a convenient tool to derive mechanistic        information.

Amino acid sequences grouped by nature of their similarity to aparticular GH family can give ideas as to the activity of the newhypothetical protein. Some of these amino acid sequences, grouped in aGH family by homology have later been suggested to have certainenzymatic activity. So, in short, grouping a new amino acid sequence ina GH family does not specifically indicate the exact enzymatic activity.The enzymatic activity must be demonstrated by an activity assay of thecloned or purified protein. If the assay is difficult determination ofthe proteins actual function can remain un-revealed for years.

Publicly available information on the GH-61 family counts presently only6 nucleotide sequences of unknown function. One document discloses,however, a guess that one of these sequences (SwissProt sequence 014405)encode an endoglucanase enzyme (Saloheimo M., Nakari-Setaelae T.,Tenkanen M., Penttilae M.; (1997) “cDNA cloning of a Trichoderma reeseicellulase and demonstration of endoglucanase activity by expression inyeast.”; Eur. J. Biochem. 249:584–591(1997). Work by the same groupconfirmed that the enzyme GH61A, when purified showed very weakcellulase activity. The group itself admitted that since the activitywas three orders of magnitude lower than normal cellulases, that perhapsthe cellulose was not the correct native substrate for the enzyme. Thegroup also made an exhaustive study of the purified enzyme with allother known carbohydrate assays (mannanase, galactanase etc.) and foundthat the enzyme had no activity for these substrates. The authorsconclude in their discussion that: “It is therefore unlikely that thefungus would produce Cel61A for its endoglucanase activity when it isalready producing more efficient endoglucanses . . . . It is possiblethat both TrCel61A and AbCel61A are active against specific parts ofmore complex natural cellulosic substrate. However, further studies areneeded to reveal the function of the glycoside hydrolase 61 enzymes”(page 6505).

Presently, the web-site of CAZY (http://afmb.cnrs-mrs.fr/CAZY/) liststhe GH-61 familiy as unclassified, meaning that properties likemechanism, catalytic nucleophile/base, catalytic proton donors, and 3-Dstructure are not known for enzymes belonging to this family. Here theonly listed known activity is endoglucanase activity.

Despite extensive screening of Trichoderma reesei recombinant yeastlibraries for cellulases and recovering many other cellulases from otherGH families we have not unambiguously identified any endoglucanasebelonging to the GH-61 family from Trichoderma reesei thus alsoindicating that GH61, if that family does include cellulases, have onlyvery weak activity and thus cannot be detected by the normally verysensistive recombinant yeast activity screening. Hence, the present 6publicly disclosed nucleotide sequences belonging the GH-61 family areeither unknown open reading frames sharing homology to SwissProtsequence O14405 or sequences cloned based on purification and sequencingof a cellulose induced gene (Isolation and characterization of acellulose-growth-specific gene from Agaricus bisporus.”; Gene119:183–190(1992), and besides the Cel61A protein mentioned above, thereis in the art no knowledge of the function and properties of any proteinor peptide belonging to the GH-61 family nor have any enzyme and/or itsfunction been reliably demonstrated.

SUMMARY OF THE INVENTION

Due to our efforts searching for new carbohydrases, we have now for thefirst time discovered polypeptides belonging to the GH-61 family whichprovides improved properties in edible products, particularly having ananti-staling effect in edible products.

Hence, in a first aspect the invention provides use of an anti-stalingGH-61 polypeptide for preparing an edible product.

In a further aspect the invention provides an isolated GH-61 polypeptidehaving an anti-staling effect in edible products.

In further aspects the invention provides a polynucleotide encoding thepolypeptide of the invention; a nucleic acid construct comprising thepolynucleotide encoding the polypeptide, operably linked to one or morecontrol sequences that direct the production of the polypeptide asuitable host; a recombinant expression vector comprising the nucleicacid construct of the invention and to a recombinant host cellcomprising the nucleic acid construct of the invention.

In a still further aspect the invention provides a compositioncomprising the GH-61 polypeptide of the invention.

In still further aspects the invention provides methods for producing aGH-61 polypeptide of the invention including a method comprising:

-   -   (a) cultivating a strain, which in its wild-type form is capable        of producing the polypeptide, to produce the polypeptide; and    -   (b) recovering the polypeptide        and a method comprising:    -   (a) cultivating a recombinant host cell of the invention under        conditions conducive for production of the polypeptide and    -   (b) recovering the polypeptide.

In a still further aspect the invention provides a transgenic plantcomprising a nucleotide sequence of the invention and capable ofexpressing the GH-61 polypeptide of the invention.

BRIEF DESCRIPTION OF DRAWINGS

No drawings

SEQUENCE LISTING

The present application contains information in the form of a sequencelisting, which is appended to the application and also submitted on adata carrier accompanying this application. The contents of the datacarrier are fully incorporated herein by reference.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term “edible product” in the context of this invention is a productprepared by heating dough, e.g. by baking or steaming, wherein saidproduct is suitable as a foodstuff for being consumed animals includingman. In particular the edible product is leavened before heating. Theproduct may be of a soft or a crisp character, either of a white, lightor dark type. Examples are steamed or baked bread (in particular white,whole-meal or rye bread), typically in the form of loaves or rolls,French baguette-type bread, pita bread, tortillas, cakes, pancakes,biscuits, cookies, pie crusts, crisp bread, steamed bread, pizza and thelike.

The terms “GH” and “GH family r homology groups” as used herein is to beunderstood as a Glycoside Hydrolases, classified in accordance with thewell established CAZY classification system.

The term “GH-61” as used herein, is to be understood as a family ofenzymes, which share common conserved sequence portions and foldings tobe classified in family 61 of the well established CAZY GHclassification system. In a preferred embodiment mature GH-61polypeptides of the invention share the following conserved portions:

-   -   H at position 1,    -   A or P at position 59,    -   G at position 60,    -   G at position 75,    -   P or A at position 76,    -   W or F at position 100,    -   F or T at position 101,    -   K or C at position 102,    -   I or V or L at position 103,    -   L or I or V or M at position 130,    -   P at position 131,    -   G, Xaa, Y at position 137–139    -   L or V or I or M at position 140    -   L or V or I or M at position 141    -   R at position 142    -   E or Q at positions 143–144,    -   L or V or I at position 148    -   H or N at position 149    -   C at position 163 and    -   P, G and P at position 209–211.

In the present context “Xaa” means any amino acid. Also in the presentcontext the numbering system for the residues is based on SEQ ID NO:2wherein the N terminal has been removed to the first histidine of thepolypeptide (there after denoted Histidine at position 1). Thisnumbering system of residues may be applied to other GH61 proteinsthrough a multiple sequence alignment with SEQ ID NO: 2 starting fromthat first Histidine, disregarding gaps generated by the multiplesequence alignment. The relative location of Histidine-1 is easilydetermined by multiple sequence alignment with SEQ ID No:2. Alignment isbe performed by using AlignX in the computer program Vector NTI ver. 7.1(Informax inc., 7600 Wisconsin Avenue, Suite #1100, Bethesda, Md. 20814,USA). The amino acid alignment is created using the Clustal W algorithm(Nucleic Acid Research, 22 (22): 4673–4680, 1994) and the followingadditional parameters: Gap opening penalty of 10, Gap extension penaltyof 0.05, Gap separation penalty range of 8. Pairwise alignmentparameters were Ktuple=1, gap penalty=3, gap length opening penalty=10,gap extension penalty=0.1, window size=5 and diagonals=5.

The term “core sequence” as used herein is to be understood as thesequence of the mature polypeptide catalytic domain excluding otherfunctional domains such as binding domains et.

The term “conserved portion” as used herein is to be understood as anamino acid or amino acid subsequence contained in all GH-61 polypeptidesof the invention and thus a common feature of all said polypeptides. Theterm conserved portions is also used for nucleotides and variantsthereof which by virtue of the degeneracy of the code encodes conservedamino acid portions.

The term “identity” as used herein, is to be understood as the homologybetween two amino acid sequences or between two nucleotide sequences.For purposes of the present invention, the degree of identity betweentwo amino acid sequences is determined by using AlignX in the program ofVector NTI ver. 7.1 (Informax inc., 7600 Wisconsin Avenue Suite #1100,Bethesda, Md. 20814, USA). Amino acid alignment is created using theClustal W algorithm (Nucleic Acid Research, 22 (22): 4673–4680,1994).The homology score matrix used in the first case was blosum62 and in thesecond case, exact identity of the amino acid sequences in the alignmentwas used. The following additional parameters were used: Gap openingpenalty of 10, Gap extension penalty of 0.05, Gap separation penaltyrange of 8. Pairwise alignment parameters were Ktuple=1, gap penalty=3,gap length opening penalty=10, gap extension penalty=0.1, window size=5and diagonals=5.

The degree of identity between two nucleotide sequences may bedetermined using the same algorithm and software package as describedabove for example with the following settings: Gap penalty of 10, andgap length penalty of 10. Pairwise alignment parameters were Ktuple=3,gap penalty=3 and windows=20.

The term “fragment” as used herein about a fragment of an polypeptide ofthe invention, is to be understood as a polypeptide having one or moreamino acids deleted from the amino and/or carboxyl terminus of the aminoacid sequence of the polypeptide, while retaining the carbohydrateactivation of the polypeptide.

The term “allelic variant” as used herein about allelic variants of apolynucleotide of the invention is to be understood as any of two ormore alternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

The term “modification(s)” as used herein about modified polypeptides ormodified polynucleotides is intended to mean any chemical modificationof the polypeptide as well as genetic manipulation of the polynucleotideencoding the polypeptide. The modification(s) can be replacement(s) ofthe amino acid side chain(s), substitution(s), deletion(s) and/orinsertions(s) in or at the amino acid(s) of interest.

The term “artificial variant” as used herein is to be understood as anmodified polypeptide capable of activating carbohydrates, which has beenproduced by an organism which is expressing a modified gene as comparedto the unmodified polynucleotide encoding the unmodified polypeptide Themodified polynucleotide (gene), from which said variant is produced whenexpressed in a suitable host, is obtained through human intervention.

The term “cDNA” as used herein is intended to mean a DNA molecule whichcan be prepared by reverse transcription from a mature, spliced, mRNAmolecule derived from a eukaryotic cell. cDNA lacks the intron sequencesthat are usually present in the corresponding genomic DNA. The initial,primary RNA transcript is a precursor to mRNA and it goes through aseries of processing events before appearing as mature spliced mRNA.These events include the removal of intron sequences by a process calledsplicing. When cDNA is derived from mRNA it therefore lacks intronsequences.

The term “nucleic acid construct” as used herein is to be understood asa nucleic acid molecule or polynucleotide, either single- ordouble-stranded, which is isolated from a naturally occurring gene orwhich has been modified to contain segments of nucleic acids in a mannerthat would not otherwise exist in nature. The term nucleic acidconstruct is synonymous with the term “expression cassette” when thenucleic acid construct contains the control sequences required forexpression of a coding sequence of the present invention.

The term “control sequence” as used herein is to be understood asnucleotide sequence including all components, which are necessary oradvantageous for the expression of a polypeptide of the presentinvention. Each control sequence may be native or foreign to thenucleotide sequence encoding the polypeptide. Such control sequencesinclude, but are not limited to, a leader, polyadenylation sequence,propeptide sequence, promoter, signal peptide sequence, andtranscription terminator. At a minimum, the control sequences include apromoter, and transcriptional and translational stop signals. Thecontrol sequences may be provided with linkers for the purpose ofintroducing specific restriction sites facilitating ligation of thecontrol sequences with the coding region of the nucleotide sequenceencoding a polypeptide.

The term “operably linked” as used herein is to be understood as aconfiguration in which a control sequence is appropriately placed at aposition relative to a nucleotide sequence encoding an polypeptide suchthat the control sequence directs the expression of the polypeptide.

The term “coding sequence” as used herein is to be understood as anucleotide sequence, which directly specifies the amino acid sequence ofthe polypeptide. The boundaries of the coding sequence are generallydetermined by an open reading frame, which usually begins with the ATGstart codon. The coding sequence typically includes DNA, cDNA, andrecombinant nucleotide sequences.

The term “expression” as used herein is to be understood as includingany step involved in the production of the polypeptide including, butnot limited to, transcription, post-transcriptional modification,translation, post-translational modification, and secretion.

The term “expression vector” as used herein is to be understood as apolynucleotide molecule, linear or circular, that comprises a segmentencoding an polypeptide of the invention, and which is operably linkedto additional segments that provide for its transcription.

The term “host cell” as used herein, includes any cell type which issusceptible to transformation with a nucleic acid construct.

The terms “polynucleotide probe”, “hybridization” as well as the variousstringency conditions are defined infra.

GH-61 Polypeptides

The GH-61 polypeptides of the invention all share the characteristicsdescribed supra. Preferably, the mature GH-61 polypeptide comprise inits naturally occurring form the conserved portions of

-   -   a Hisitidine (H) at position 1,    -   A or P at position 59,    -   G at position 60,    -   G at position 75,    -   P or A at position 76,    -   W or F at position 100,    -   F or T at position 101,    -   K or C at position 102,    -   I or V or L at position 103,    -   L or I or V or M at position 130,    -   P at position 131,    -   G, Xaa and Y at position 137–139    -   L or V or I or M at position 140,    -   L or V or I or M at position 141,    -   R at position 142,    -   E or Q at positions 143–144,    -   L or V or I at position 148,    -   H or N at position 149,    -   C at position 163 and    -   P and G and P at position 209–211,    -   when adopting the above mentioned numbering system.

In particular the GH-61 polypeptide is an isolated polypeptide meaningthat a preparation of the polypeptide contains at the most 90% by weightof other polypeptide material with which it may be natively associated(lower percentages of other polypeptide material are preferred, e.g. atthe most 80% by weight, at the most 60% by weight, at the most 50% byweight, at the most 40% at the most 30% by weight, at the most 20% byweight, at the most 10% by weight, at the most 9% by weight, at the most8% by weight, at the most 6% by weight, at the most 5% by weight, at themost 4% at the most 3% by weight, at the most 2% by weight, at the most1% by weight and at the most ½% by weight). Thus, in particular theisolated polypeptide is at least 92% pure, i.e. that the polypeptideconstitutes at least 92% by weight of the total polypeptide materialpresent in the preparation, and higher percentages are preferred such asat least 94% pure, at least 95% pure, at least 96% pure, at least 96%pure, at least 97% pure, at least 98% pure, at least 99%, and at themost 99.5% pure. In particular, it is preferred that the polypeptidesdisclosed herein are in “essentially pure form”, i.e. that thepolypeptide preparation is essentially free of other polypeptidematerial with which it is natively associated. This can be accomplished,for example, by preparing the polypeptide by means of well-knownrecombinant methods.

The GH-61 polypeptide of the invention may be synthetically made,naturally occurring or a combination thereof. In a particular embodimentthe polypeptide of the invention may be obtained from a microorganismsuch as a prokaryotic cell, an archaeal cell or a eucaryotic cell. Thecell may further have been modified by genetic engineering (cf. Sourcesof GH-61 polypeptides, vide infra).

In another particular embodiment, the polypeptide of the invention havesize from about 5 kDa to about 500 kDa, in particular from about 10 kDato about 250 kDa, more particularly from about 20 kDa to 100 kDa.

In a still further embodiment, the polypeptide of the invention may befunctionally stabile over at a temperature of up to 120° C., inparticular up to 100° C. in particular up to 80° C., more particularlyup to 60° C.

The polypeptides of the invention are as said GH-61 polypeptides andhave an anti-staling effect on edible products of the invention. Inparticular the GH-61 polypeptides have a beneficial effect on thefirmness, the elasticity and/or the water mobility of the edibleproduct.

The advantageous effects in edible products prepared using the GH-61polypeptides of the invention are clear from the experiments. Furtherdue to its classification in the GH-61 family it is presentlycontemplated that the GH-61 polypeptide of the invention is an enzyme,in particular a having hydrolase activity, particularly a carbohydraseactivity. Further, concluding from the experiments disclosed herein theexemplified GH-61 polypeptides seems to have at least a minor activitytowards oat xylan, birchwood xylan and/or wheat arabino-xylan. Hence, ina particular embodiment the GH-61 polypeptide of the invention exhibitsat least a minor activity against these substrates.

In a further embodiment, the GH-61 polypeptide of the invention mayexhibit optimum substrate hydrolysis at pH 5–9.

In a still further embodiment, the polypeptide of the invention mayexhibit optimum substrate hydrolysis at a temperature within the rangefrom about 10° C. to about 90° C., such as about 10° C. to about 80° C.,particularly in the range from about 20° C. to about 60° C. or about 70°C. to about 90° C.

In a particular embodiment, the polypeptide exhibit at least 20%, inparticular at least 40%, such as at least 50%, in particular at least60%, such as at least 70%, more particularly at least 80%, such as atleast 90%, most particularly at least 95%, such as about or at least100% of the polypeptide activity of either of the polypeptidesconsisting of the amino acid sequences shown as the mature GH-61polypeptide of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6. The polypeptidepreferably comprises or consists of, an amino acid sequence which has adegree of identity to the amino acids of the mature GH-61 polypeptidesof SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6 of at least 65%, particularlyat least 70%, e.g. at least 75%, more particularly at least 80%, such asat least 85%, even more particularly at least 90%, most particularly atleast 95%, e.g. at least 96%, such as at least 97%, and even mostparticularly at least 98%, such as at least 99% (hereinafter “homologouspolypeptides”), with the proviso that the polypeptide belong to theGH-61 family and that the polypeptide preferably comprise the abovementioned conserved amino acid portions.

In a particular embodiment, the amino acid sequence differs by at themost ten amino acids (e.g. by ten amino acids), in particular by at themost five amino acids (e.g. by five amino acids), such as by at the mostfour amino acids (e.g. by four amino acids), e.g. by at the most threeamino acids (e.g. by three amino acids) from amino acids of the matureGH-61 polypeptides of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, observingthe above mentioned proviso. In a particular embodiment, the amino acidsequence of the polypeptide differs by at the most two amino acids (e.g.by two amino acids), such as by one amino acid from amino acids of themature GH-61 polypeptides of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6.

Aligning the polypeptides consisting of the amino acid sequence shown asamino acids of the mature GH-61 polypeptides of SEQ ID NO:2, SEQ ID NO:4or SEQ ID NO:6 with the closest prior art and using the aligment methoddescribed above (see the section entitled “Definitions”), the closestknown sequence is Cel61A a Trichoderma reesei (Hypocrea jecorina)endo-1,4-glucanase IV.

Particularly, the polypeptide comprises the amino acid sequence of themature GH-61 polypeptides of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6;an allelic variant thereof; or a fragment thereof that provides ananti-staling effect on edible products

In a further particular embodiment, the polypeptide consists of aminoacids of the mature GH-61 polypeptides of SEQ ID NO:2, SEQ ID NO:4 orSEQ ID NO:6

The polypeptide of the invention may be a wild-type polypeptideidentified and isolated from a natural source.

Furthermore, the polypeptide of the invention may be prepared by the DNAshuffling technique, such as described in J. E. Ness et al. NatureBiotechnology 17, 893–896 (1999).

The present invention also encompasses artificial variants of thepolypeptides of the invention, where said polypeptides has been mutatedby adding, substituting and/or deleting one or more amino acids fromsaid polypeptide polypeptides while retaining the polypeptide activityand the conserved portions. Hence, the polypeptide of the invention maybe an artificial variant of a GH-61 polypeptide, wherein at least onesubstitution, deletion and/or insertion of an amino acid has been madeto the amino acid sequence in a parent GH-61 polypeptide amino acidsequence. In particular the artificial variant comprises, in particularconsists of, an amino acid sequence that has at least one substitution,deletion and/or insertion of an amino acid as compared to amino acidsthe mature GH-61 polypeptides of SEQ ID NO:2, SEQ ID NO:4 of SEQ IDNO:6. In particular the mutation is made outside the conserved portionof the sequence or a substitution may be made within a conserved portionwhile maintaining the polypeptide in the GH-61 family. For example forposition 76 P may be substituted by A, while sill being conservedaccording to the definition.

Such artificial variants may be constructed by standard techniques knownin the art usually followed by screening and/or characterization.Standard techniques includes classical mutagenesis, e.g. by UVirradiation of the cells or treatment of cells with chemical mutagens asdescribed by Gerhardt et al. (1994); in vivo gene shuffling as describedin WO 97/07205; in vitro shuffling as described by Stemmer, (1994) or WO95/17413, random mutagenesis as described by Eisenstadt E. et al.,(1994); PCR techniques as described by Poulsen et al. (1991); familyshuffling as described by J. E. Ness, et al, Nature Biotechnology,vol.17, pp. 893–896 (1999); site-directed mutagenesis as described bySambrook et al. (1989), Sambrook et al., Molecular Cloning. A LaboratoryManual, Cold Spring Harbor, N.Y. A general description of nucleotidesubstitution can be found in e.g. Ford et al., 1991, Protein Expressionand Purification 2, p. 95–107.

Such standard genetic engineering methods may also be used prepare adiversified library of variant nucleotide sequences from the genesencoding one or more parent polypeptides, expressing the polypeptidevariants in a suitable host cell and selecting a preferred variant(s). Adiversified library can be established by a range of techniques known tothe art (Reetz M T; Jaeger K E, in Biocatalysis—from Discovery toApplication edited by Fessner W D, Vol. 200, pp. 31–57 (1999); Stemmer,Nature, vol. 370, p.389–391, 1994; Zhao and Arnold, Proc. Natl. Acad.Sci., USA, vol. 94, pp. 7997–8000, 1997; or Yano et al., Proc. Natl.Acad. Sci., USA, vol. 95, pp 5511–5515, 1998).

In one embodiment of the invention, amino acid changes (in theartificial variant as well as in wild-type polypeptides) are of a minornature, that is conservative amino acid substitutions that do notsignificantly affect the folding and/or activity of the protein; smalldeletions, typically of one to about 30 amino acids; small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small linker peptide of up to about 20–25 residues; or asmall extension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine, valine andmethionine), aromatic amino acids (phenylalanine, tryptophan andtyrosine), and small amino acids (glycine, alanine, serine andthreonine). Amino acid substitutions which do not generally alter and orimpair the function of a protein are known in the art and are described,for example, by H. Neurath and R. L. Hill, 1979, In, The Proteins,Academic Press, New York. The most commonly occurring exchanges areAla/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val,Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/lle, Leu/Val, Ala/Glu,and Asp/Gly as well as these in reverse.

In an interesting embodiment of the invention, the amino acid changesare of such a nature that the physico-chemical properties of thepolypeptides are altered. For example, amino acid changes may beperformed, which improve the thermal stability of the polypeptide, whichalter the substrate specificity, which changes the pH optimum, and thelike.

Particularly, the number of such substitutions, deletions and/orinsertions in the polypeptide of the invention, particularly those ofthe mature polypeptides of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6 toproduce an artificial variant is at the most 10, such as at the most 9,e.g. at the most 8, more preferably at the most 7, e.g. at the most 6,such as at the most 5, most preferably at the most 4, e.g. at the most3, such as at the most 2, in particular at the most 1.

In a particular embodiment, the artificial variant is a variant, whichhas an altered, preferably reduced, immunogenicity, especiallyallergenicity, in animals including man as compared to a parentpolypeptide. The term “immunogenicity” in this context is to beunderstood as the artificial variant having an altered, in particularreduced, binding ability to antibodies as well as having an alteredability to provoke production of antibodies when administered to ananimal, including intravenous, cutaneous, subcutaneous, oral andintratracheal administration.

Administration of the artificial variant may causes an alteration in theimmunoglobolin levels in the animal body, such as in IgE, IgG and IgM oran alteration in the cytokine level in the animal body. Methods formapping immunogenic/antigenic epitopes of a protein, preparing variantswith altered immunogenicity and methods for measuring an immunologicalresponse is well known to the art and are described e.g. in WO 92/10755,WO 00/26230, WO 00/26354 and WO 01/31989.

In a further embodiment, the present invention relates to GH-61polypeptides having an anti-staling effect in edible products which areencoded by nucleotide sequences which hybridize under very lowstringency conditions, particularly under low stringency conditions,more particularly under medium stringency conditions, more particularlyunder medium-high stringency conditions, even more particularly underhigh stringency conditions, and most particularly under very highstringency conditions with a polynucleotide probe selected from thegroup consisting of

-   -   (i) the complementary strand of nucleotides 52 to 699 of SEQ ID        NO:1, 46 to 957 of SEQ ID NO:3 or 58 to 660 of SEQ ID NO:5,    -   (ii) the complementary strand of the cDNA sequence contained in        nucleotides 52 to 699 of SEQ ID NO:1, 46 to 957 of SEQ ID NO:3        or 58 to 660 of SEQ ID NO:5    -   (iii) the complementary strand of nucleotides 46 to 857 of SEQ        ID NO:3,    -   (iv) the complementary strand of nucleotides 52 to 300 of SEQ ID        NO:1, 46 to 501 of SEQ ID NO:3 or 58 to 300 of SEQ ID NO:5        and/or    -   (v) the complementary strand of nucleotides 301 to 699 of SEQ ID        NO:1, 502 to 957 of SEQ ID NO:3 or 301 to 660 of SEQ ID NO:5,        (J. Sambrook, E. F. Fritsch, and T. Maniatus, 1989, Molecular        Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor,        N.Y.).

In particular, the polypeptide of the invention is encoded by apolynucleotide comprising the nucleotide sequence of nucleotides 52 to699 of SEQ ID NO:1, 46 to 957 of SEQ ID NO:3 or 58 to 660 of SEQ ID NO:5or sequences differing from 52 to 699 of SEQ ID NO:1, 46 to 957 of SEQID NO:3 or 58 to 660 of SEQ ID NO:5 by virtue of the degeneracy of thegenetic code. More particularly, the polypeptide of the invention isencoded by a polynucleotide consisting of nucleotide sequence ofnucleotides 52 to 699 of SEQ ID NO:1, 46 to 957 of SEQ ID NO:3 or 58 to660 of SEQ ID NO:5 or sequences differing from 52 to 699 of SEQ ID NO:1,46 to 957 of SEQ ID NO:3 or 58 to 660 of SEQ ID NO:5 by virtue of thedegeneracy of the genetic code.

The nucleotide sequences of nucleotides SEQ ID NO:1, SEQ ID NO:3 or ofSEQ ID NO:5 or a subsequence thereof, as well as the amino acidsequences of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6 or a fragmentthereof, may be used to design a polynucleotide probe to identify andclone DNA encoding GH-61 polypeptides of the invention from strains ofdifferent genera or species according to methods well known in the art.In particular, such probes can be used for hybridization with thegenomic or cDNA of the genus or species of interest, following standardSouthern blotting procedures, in order to identify and isolate thecorresponding gene therein. Such probes can be considerably shorter thanthe entire sequence, but should be at least 15, preferably at least 25,more preferably at least 35 nucleotides in length, such as at least 70nucleotides in length. It is, however, preferred that the polynucleotideprobe is at least 100 nucleotides in length. For example, thepolynucleotide probe may be at least 200 nucleotides in length, at least300 nucleotides in length, at least 400 nucleotides in length or atleast 500 nucleotides in length. Even longer probes may be used, e.g.,polynucleotide probes which are at least 600 nucleotides in length, atleast 700 nucleotides in length, at least 800 nucleotides in length, orat least 900 nucleotides in length. Both DNA and RNA probes can be used.The probes are typically labeled for detecting the corresponding gene(for example, with ³²P, ³H, 35S, biotin, or avidin).

Thus, a genomic DNA or cDNA library prepared from such other organismsmay be screened for DNA which hybridizes with the probes described aboveand which encodes the polypeptides of the invention. Genomic or otherDNA from such other organisms may be separated by agarose orpolyacrylamide gel electrophoresis, or other separation techniques. DNAfrom the libraries or the separated DNA may be transferred to, andimmobilized, on nitrocellulose or other suitable carrier materials. Inorder to identify a clone or DNA which has the required homology and/oridentity or is homologous and/or identical with of nucleotides 52 to 699of SEQ ID NO:1, 46 to 957 of SEQ ID NO:3 or 58 to 660 of SEQ ID NO:5 thecarrier material with the immobilized DNA is used in a Southern blot.

For purposes of the present invention, hybridization indicates that thenucleotide sequence hybridizes to a labeled polynucleotide probe whichagain hybridizes to the nucleotide sequence shown in nucleotides 52 to699 of SEQ ID NO:1, 46 to 957 of SEQ ID NO:3 or 58 to 660 of SEQ ID NO:5under very low to very high stringency conditions. Molecules to whichthe polynucleotide probe hybridizes under these conditions may bedetected using X-ray film or by any other method known in the art.Whenever the term “polynucleotide probe” is used in the present context,it is to be understood that such a probe contains at least 15nucleotides. In particular the probe comprise nucleotides encodingconserved portions of a Hisitidine (H) at position 1, A or P at position59, G at position 60, G at position 75, P or A at position 76, W or F atposition 100, F or T at position 101, K or C at position 102, I or V orL at position 103, L or I or V or M at position 130, P at position 131,G and Xaa and Y at position 137–139, L or V or I or M at position 140, Lor V or I or M at position 141, R at position 142, E or Q at positions143–144, L or V or I at position 148, H or N at position 149, C atposition 163 and P and G and P at position 209–211 in the maturepolypeptide.

In an interesting embodiment, the polynucleotide probe is thecomplementary strand of nucleotides 52 to 300 of SEQ ID NO:1, 46 to 501of SEQ ID NO:3 or 58 to 300 of SEQ ID NO:5. In another embodiment thepolynucleotide probe is the complementary strand of nucleotides 301 to699 of SEQ ID NO:1, 502 to 957 of SEQ ID NO:3 or 301 to 660 of SEQ IDNO:5.

In another interesting embodiment, the polynucleotide probe is thecomplementary strand of the nucleotide sequence which encodes thepolypeptide of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6 or the maturepolypeptides thereof. In a further interesting embodiment, thepolynucleotide probe is the complementary strand of SEQ ID NO:1, SEQ IDNO:3 or SEQ ID NO:5. In a still further interesting embodiment, thepolynucleotide probe is the complementary strand of the region of SEQ IDNO:1, SEQ ID NO:3 or SEQ ID NO:5 encoding the mature polypeptide.

For long probes of at least 100 nucleotides in length, very low to veryhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 533 SSPE, 1.0% SDS, 5× Denhardt's solution,100 μg/ml sheared and denatured salmon sperm DNA, following standardSouthern blotting procedures. Preferably, the long probes of at least100 nucleotides do not contain more than 1000 nucleotides. For longprobes of at least 100 nucleotides in length, the carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.1% SDS at42° C. (very low stringency), preferably washed three times each for 15minutes using 0.5 ×SSC, 0.1% SDS at 42° C. (low stringency), morepreferably washed three times each for 15 minutes using 0.2×SSC, 0.1%SDS at 42° C. (medium stringency), even more preferably washed threetimes each for 15 minutes using 0.2×SSC, 0.1% SDS at 55° C. (medium-highstringency), most preferably washed three times each for 15 minutesusing 0.1×SSC, 0.1% SDS at 60° C. (high stringency), in particularwashed three times each for 15 minutes using 0.1×SSC, 0.1% SDS at 68° C.(very high stringency).

Although not particularly preferred, it is contemplated that shorterprobes, e.g. probes which are from about 15 to 99 nucleotides in length,such as from about 15 to about 70 nucleotides in length, may be also beused. For such short probes, stringency conditions are defined asprehybridization, hybridization, and washing post-hybridization at 5° C.to 10° C. below the calculated Tm using the calculation according toBolton and McCarthy (1962, Proceedings of the National Academy ofSciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA,0.5% NP-40, 1× Denhardt's solution, 1 mM sodium pyrophosphate, 1 mMsodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per mlfollowing standard Southern blotting procedures.

For short probes which are about 15 nucleotides to 99 nucleotides inlength, the carrier material is washed once in 6×SCC plus 0.1% SDS for15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10° C.below the calculated Tm.

Sources of Polypeptides

The polypeptide of the present invention may be obtained frommicroorganisms of any genus. For purposes of the present invention, theterm “obtained from”, as used herein shall mean that the polypeptideencoded by the nucleotide sequence is produced by a cell in which thenucleotide sequence is naturally present or into which the nucleotidesequence has been inserted.

Further polypeptides obtainable from microorganisms are in a particularembodiment an extracellular polypeptides i.e. a polypeptide which issecreted or otherwise exported from a microorganism to its surroundingmedium.

Eukaryotic Sources

The polypeptide of the invention may be obtained from eukaryotesparticularly, plant cells or fungi. Particularly, the polypeptide may bederived from fungi that degrade carbohydrates, such as cellulosicsubstrates. Such fungi include e.g. Ascomycota, Basidiomycota,Zygomycota or Oomycota. In particular Verticillium tenerum, Coprinuscinerius, Diplodia gossypinna, Humicola insolens, Dichotomocladiumhesseltinei, Pseudoplectania nigrella, Psilocybe inquilina andTrichophaea saccata.

Other relevant fungi may be yeasts such as a Candida, Kluyveromyces,Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia or filamentousfungi such as an Acremonium, Aspergillus, Aureobasidium, Cryptococcus,Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora,Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, orTrichoderma.

In an interesting embodiment, the polypeptide is derived fromSaccharomyces carisbergensis, Saccharomyces cerevisiae, Saccharomycesdiastaticus, Saccharomyces douglasii, Saccharomyces kluyveri,Saccharomyces norbensis or Saccharomyces oviformis.

In another interesting embodiment, the polypeptide is derived fromAspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus,Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger,Aspergillus oryzae, Fusarium bactridioides, Fusarium cerealis, Fusariumcrookwellense, Fusarium culmorum, Fusarium graminearum, Fusariumgraminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum,Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusariumsarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusariumtorulosum, Fusarium trichothecioides, Fusarium venenatum, Humicolainsolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila,Neurospora crassa, Penicillium purpurogenum, Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,or Trichoderma viride.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

Furthermore, such polypeptides may be identified and obtained from othersources including microorganisms isolated from nature (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms from natural habitats are well known in theart. The nucleotide sequence may then be derived by similarly screeninga genomic or cDNA library of another microorganism. Once a nucleotidesequence encoding a polypeptide has been detected with the probe(s), thesequence may be isolated or cloned by utilizing techniques which areknown to those of ordinary skill in the art (see, e.g., Sambrook et al.,1989, supra).

Polypeptides encoded by nucleotide sequences of the present inventionalso include fused polypeptides or cleavable fusion polypeptides inwhich another polypeptide is fused at the N-terminus or the C-terminusof the polypeptide or fragment thereof. A fused polypeptide is producedby fusing a nucleotide sequence (or a portion thereof) encoding anotherpolypeptide to a nucleotide sequence (or a portion thereof) of thepresent invention. Techniques for producing fusion polypeptides areknown in the art, and include ligating the coding sequences encoding thepolypeptides so that they are in frame and that expression of the fusedpolypeptide is under control of the same promoter(s) and terminator.

Nucleotide Sequences

The present invention also relates to polynucleotides comprising anucleotide sequence, which encodes a GH-61 polypeptide of the invention.In a particular embodiment, the nucleotide sequence is set forth in SEQID NO:1, SEQ ID NO:3 or SEQ ID NO:5. In a more particular embodiment,the nucleotide sequence is the region of SEQ ID NO:1, SEQ ID NO:3 or SEQID NO:5 encoding the mature GH-61 polypeptide.

The present invention also encompasses polynucleotides comprising,particularly containing or more particularly consisting of, nucleotidesequences which encode a polypeptide consisting of the amino acidsequence of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6. or the maturepolypeptide thereof including nucleotide sequences differing from SEQ IDNO:1, SEQ ID NO:3 or SEQ ID NO:5 by virtue of the degeneracy of thegenetic code.

The present invention also relates to polynucleotides comprising,preferably consisting of, a subsequence:

-   -   which encode fragments of SEQ ID NO:2 that have an anti-staling        effect in edible products and contains the conserved portions.        In particular the subsequence is a subsequence of SEQ ID NO:1    -   which encode fragments of SEQ ID NO:4 that have an anti-staling        effect in edible products and contains the conserved portions.        In particular the subsequence is a subsequence of SEQ ID NO:3    -   which encode fragments of SEQ ID NO:6 that have an anti-staling        effect in edible products and contains the conserved portions.        In particular the subsequence is a subsequence of SEQ ID NO:5

A subsequence of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5 is a nucleotidesequence encompassed by SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5 exceptthat one or more nucleotides from the 5′ and/or 3′ end have beendeleted.

The present invention also relates to polynucleotides comprising,preferably consisting of, a modified nucleotide sequence which comprisesat least one modification/mutation in the mature polypeptide codingsequence of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5, and where themodified nucleotide sequence encodes a polypeptide comprising a H atposition 1, A or P at position 59, G at position 60, G at position 75, Por A at position 76, W or F at position 100, F or T at position 101, Kor C at position 102I or V or L at position 103, L or I or V or M atposition 130, P at position 131, G and Xaa and Y at position 137–139, Lor V or I or M at position 140, L or V or I or M at position 141, R atposition 142, E or Q at positions 143–144, L or V or I at position 148,H or N at position 149, C at position 163 and P and G and P at position209–211.

The techniques used to isolate and/or clone a nucleotide sequenceencoding an polypeptide are known in the art and include isolation fromgenomic DNA, preparation from cDNA, or a combination thereof. Thecloning of the nucleotide sequences of the present invention from suchgenomic DNA can be effected, e.g., by using the well known polymerasechain reaction (PCR) or antibody screening of expression libraries todetect cloned DNA fragments with shared structural features. See, e.g.,Innis et al., 1990, PCR: A Guide to Methods and Application, AcademicPress, New York. Other amplification procedures such as ligase chainreaction (LCR), ligated activated transcription (LAT) and nucleotidesequence-based amplification (NASBA) may be used. The nucleotidesequence may be cloned from a strain of Humicola or Coprinus, or anotheror related organism and thus, for example, may be an allelic or speciesvariant of the polypeptide encoding region of the nucleotide sequence.

The nucleotide sequence may be obtained by standard cloning proceduresused in genetic engineering to relocate the nucleotide sequence from itsnatural location to a different site where it will be reproduced. Thecloning procedures may involve excision and isolation of a desiredfragment comprising the nucleotide sequence encoding the polypeptide,insertion of the fragment into a vector molecule, and incorporation ofthe recombinant vector into a host cell where multiple copies or clonesof the nucleotide sequence will be replicated. The nucleotide sequencemay be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or anycombinations thereof.

The present invention also relates to a polynucleotide encoding a GH-61polypeptide of the invention comprising, preferably consisting of, anucleotide sequence which has at least 65% identity with nucleotides 52to 699 of SEQ ID NO:1, 46 to 957 of SEQ ID NO:3 or 58 to 660 of SEQ IDNO:5. Particularly, the nucleotide sequence has at least 70% identity,e.g. at least 80% identity, such as at least 90% identity, morepreferably at least 95% identity, such as at least 96% identity, e.g. atleast 97% identity, even more preferably at least 98% identity, such asat least 99% with nucleotides 52 to 699 of SEQ ID NO:1, 46 to 957 of SEQID NO:3 or 58 to 660 of SEQ ID NO:5. The degree of identity between twonucleotide sequences is determined as described previously (see thesection entitled “Definitions”). Particularly, the nucleotide sequencecomprises nucleotides 52 to 699 of SEQ ID NO:1, 46 to 957 of SEQ ID NO:3or 58 to 660 of SEQ ID NO:5. In an even more particular embodiment, thenucleotide sequence consists of nucleotides 52 to 699 of SEQ ID NO:1, 46to 957 of SEQ ID NO:3 or 58 to 660 of SEQ ID NO:5.

Modification of a nucleotide sequence encoding an polypeptide of thepresent invention may be necessary for the synthesis of an polypeptide,which comprises an amino acid sequence that has at least onesubstitution, deletion and/or insertion as compared to amino acids18–233 of SEQ ID NO:2, 16–319 of SEQ ID NO:4 or 20–220 of SEQ ID NO:6.These artificial variants may differ in properties or other way from thepolypeptide isolated from its native source, e.g., variants may differin carbohydrate activation capabilities, carbohydrate specificity,thermostability, pH optimum or the like.

It will be apparent to those skilled in the art that such modificationscan be made to preserve the membership of the polypeptide to the GH-61homology group and can be made outside regions critical to the functionof the molecule regions and still result in a polypeptide. Amino acidresidues essential to the function or the GH-61 characteristics of thepolypeptide encoded by the nucleotide sequence of the invention aretherefore preferably not subject to modification, such as substitution.Amino acid residues essential to the function may be identifiedaccording to procedures known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (see, e.g., Cunningham andWells, 1989, Science 244: 1081–1085). In the latter technique, mutationsare introduced at every positively charged residue in the molecule, andthe resultant mutant molecules are tested for the carbohydrateactivation function to identify amino acid residues that are critical tothe function of the molecule. Sites of carbohydrate-polypeptideinteraction can also be determined by analysis of the three-dimensionalstructure as determined by such techniques as nuclear magnetic resonanceanalysis, crystallography or photoaffinity labelling (see, e.g., de Voset al., 1992, Science 255: 306–312; Smith et al., 1992, Journal ofMolecular Biology 224: 899–904; Wlodaver et al.,1992, FEBS Letters 309:59–64).

Moreover, a nucleotide sequence encoding a polypeptide of the presentinvention may be modified by introduction of nucleotide substitutionswhich do not give rise to another amino acid sequence of the polypeptideencoded by the nucleotide sequence, but which correspond to the codonusage of the host organism intended for production of the polypeptide.

The introduction of a mutation into the nucleotide sequence to exchangeone nucleotide for another nucleotide may be accomplished bysite-directed mutagenesis using any of the methods known in the art.Particularly useful is the procedure, which utilizes a supercoiled,double stranded DNA vector with an insert of interest and two syntheticprimers containing the desired mutation. The oligonucleotide primers,each complementary to opposite strands of the vector, extend duringtemperature cycling by means of Pfu DNA polymerase. On incorporation ofthe primers, a mutated plasmid containing staggered nicks is generated.Following temperature cycling, the product is treated with Dpnl which isspecific for methylated and hemimethylated DNA to digest the parentalDNA template and to select for mutation-containing synthesized DNA.Other procedures known in the art may also be used. For a generaldescription of nucleotide substitution, see, e.g. Ford et al., 1991,Protein Expression and Purification 2: 95–107.

The present invention also relates to a polynucleotide comprising,preferably consisting of, a nucleotide sequence which encodes a GH-61polypeptide and which hybridizes under very low stringency conditions,preferably under low stringency conditions, more preferably under mediumstringency conditions, more preferably under medium-high stringencyconditions, even more preferably under high stringency conditions, andmost preferably under very high stringency conditions with apolynucleotide probe selected from the group consisting of:

-   -   (i) the complementary strand of nucleotides 52 to 699 of SEQ ID        NO: 1, 46 to 957 of SEQ ID NO:3 or 58 to 660 of SEQ ID NO:5,    -   (ii) the complementary strand of the cDNA sequence contained in        nucleotides 52 to 699 of SEQ ID NO:1, 46 to 957 of SEQ ID NO:3        or 58 to 660 of SEQ ID NO:5    -   (iii) the complementary strand of nucleotides 46 to 857 of SEQ        ID NO:3,    -   (iv) the complementary strand of nucleotides 52 to 300 of SEQ ID        NO:1, 46 to 501 of SEQ ID NO:3 or 58 to 300 of SEQ ID NO:5,    -   (v) the complementary strand of nucleotides 301 to 699 of SEQ ID        NO:1, 502 to 957 of SEQ ID NO:3 or 301 to 660 of SEQ ID NO:5,        (J. Sambrook, E. F. Fritsch, and T. Maniatus, 1989, Molecular        Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor,        N.Y.).

As will be understood, details and particulars concerning hybridizationof the nucleotide sequences will be the same or analogous to thehybridization aspects discussed in the section titled “GH-61polypeptides” herein.

The present invention also encompass a storage medium, such as acomputer disk, containing in digital form or in any other form thesequences as mentioned herein.

Nucleic Acid Construct Comprising Nucleotide Sequences

The present invention also relates to nucleic acid constructs comprisinga nucleotide sequence of the invention operably linked to one or morecontrol sequences that direct the expression of the coding sequence in asuitable host cell under conditions compatible with the controlsequences.

A nucleotide sequence encoding a polypeptide of the invention may bemanipulated in a variety of ways to provide for expression of thepolypeptide. Manipulation of the nucleotide sequence prior to itsinsertion into a vector may be desirable or necessary depending on theexpression vector. The techniques for modifying nucleotide sequencesutilizing recombinant DNA methods are well known in the art.

The control sequence may be an appropriate promoter sequence, anucleotide sequence which is recognized by a host cell for expression ofthe nucleotide sequence. The promoter sequence contains transcriptionalcontrol sequences, which mediate the expression of the polypeptide. Thepromoter may be any nucleotide sequence which shows transcriptionalactivity in the host cell of choice including mutant, truncated, andhybrid promoters, and may be obtained from genes encoding extracellularor intracellular polypeptides either homologous or heterologous to thehost cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention, especially in abacterial host cell, are the promoters obtained from the E. coli lacoperon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilislevansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus amyloliquefaciens alphaamylase gene (amyQ), Bacilluslicheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylBgenes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978,Proceedings of the National Academy of Sciences USA 75: 3727–3731), aswell as the tac promoter (DeBoer et al., 1983, Proceedings of theNational Academy of Sciences USA 80: 21–25). Further promoters aredescribed in “Useful proteins from recombinant bacteria” in ScientificAmerican, 1980, 242: 74–94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus oryzaeTAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, andFusarium oxysporum trypsin-like protease (WO 96/00787), as well as theNA2-tpi promoter (a hybrid of the promoters from the genes forAspergillus niger neutral alpha-amylase and Aspergillus oryzae triosephosphate isomerase), and mutant, truncated, and hybrid promotersthereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), andSaccharomyces cerevisiae 3-phosphoglycerate kinase. Other usefulpromoters for yeast host cells are described by Romanos et al., 1992,Yeast 8: 423–488.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleotide sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus oryzae TAKA amylase, Aspergillus nigerglucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillusniger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anon-translated region of an mRNA which is important for translation bythe host cell. The leader sequence is operably linked to the 5′ terminusof the nucleotide sequence encoding the polypeptide. Any leader sequencethat is functional in the host cell of choice may be used in the presentinvention. Preferred leaders for filamentous fungal host cells areobtained from the genes for Aspergillus oryzae TAKA amylase andAspergillus nidulans triose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleotide sequence and which,when transcribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencewhich is functional in the host cell of choice may be used in thepresent invention.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Molecular Cellular Biology 15: 5983–5990.

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleotidesequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion which encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region whichis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not naturallycontain a signal peptide coding region. Alternatively, the foreignsignal peptide coding region may simply replace the natural signalpeptide coding region in order to enhance secretion of the polypeptide.However, any signal peptide coding region which directs the expressedpolypeptide into the secretory pathway of a host cell of choice may beused in the present invention.

The signal peptide coding region is nucleotides 1 to 51 of SEQ ID NO:1,1 to 51 of SEQ ID NO:3 or 1 to 57 of SEQ ID NO:5 which encode aminoacids 18 to 233 of SEQ ID NO:2, 16 to 319 of SEQ ID NO:4 or 20 to 220 ofSEQ ID NO:6.

Effective signal peptide coding regions for bacterial host cells are thesignal peptide coding regions obtained from the genes for Bacillus NCIB11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase,Bacillus licheniformis subtilisin, Bacillus licheniformis betalactamase,Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), andBacillus subtilis prsA. Further signal peptides are described by Simonenand Palva, 1993, Microbiological Reviews 57: 109–137.

Effective signal peptide coding regions for filamentous fungal hostcells are the signal peptide coding regions obtained from the genes forAspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, and Humicola lanuginosa lipase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding regions are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide may be denoted a pro-polypeptideor propolypeptide. A propolypeptide is generally inactive and can beconverted to a mature active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding region may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei asparticproteinase, and Myceliophthora thermophila laccase (WO 95/33836).

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

In yeast, the ADH2 system or GAL1 system may be used. In filamentousfungi, the TAKA alpha-amylase promoter, Aspergillus niger glucoamylasepromoter, and Aspergillus oryzae glucoamylase promoter may be used asregulatory sequences. Other examples of regulatory sequences are thosewhich allow for gene amplification. In eukaryotic systems, these includethe dihydrofolate reductase gene which is amplified in the presence ofmethotrexate, and the metallothionein genes which are amplified withheavy metals. In these cases, the nucleotide sequence encoding thepolypeptide would be operably linked with the regulatory sequence.

Recombinant Expression Vector Comprising Nucleic Acid Construct

The present invention also relates to recombinant expression vectorscomprising the nucleic acid construct of the invention. The variousnucleotide and control sequences described above may be joined togetherto produce a recombinant expression vector, which may include one ormore convenient restriction sites to allow for insertion or substitutionof the nucleotide sequence encoding the polypeptide at such sites.Alternatively, the nucleotide sequence of the present invention may beexpressed by inserting the nucleotide sequence or a nucleic acidconstruct comprising the sequence into an appropriate vector forexpression. In creating the expression vector, the coding sequence islocated in the vector so that the coding sequence is operably linkedwith the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) which can be conveniently subjected to recombinant DNA proceduresand can bring about the expression of the nucleotide sequence. Thechoice of the vector will typically depend on the compatibility of thevector with the host cell into which the vector is to be introduced. Thevectors may be linear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e. a vectorwhich exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g. a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.

The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The vectors of the present invention preferably contain one or moreselectable markers which permit easy selection of transformed cells. Aselectable marker is a gene the product of which provides for biocide orviral resistance, resistance to heavy metals, prototrophy to auxotrophs,and the like.

Examples of bacterial selectable markers are the dal genes from Bacillussubtilis or Bacillus licheniformis, or markers which confer antibioticresistance such as ampicillin, kanamycin, chloramphenicol ortetracycline resistance. Suitable markers for yeast host cells are ADE2,HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in afilamentous fungal host cell include, but are not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hygB (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),trpC (anthranilate synthase), as well as equivalents thereof.

Preferred for use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The vectors of the present invention preferably contain an element(s)that permits stable integration of the vector into the host cell'sgenome or autonomous replication of the vector in the cell independentof the genome.

For integration into the host cell genome, the vector may rely on thenucleotide sequence encoding the polypeptide or any other element of thevector for stable integration of the vector into the genome byhomologous or nonhomologous recombination. Alternatively, the vector maycontain additional nucleotide sequences for directing integration byhomologous recombination into the genome of the host cell. Theadditional nucleotide sequences enable the vector to be integrated intothe host cell genome at a precise location(s) in the chromosome(s). Toincrease the likelihood of integration at a precise location, theintegrational elements should preferably contain a sufficient number ofnucleotides, such as 100 to 1,500 base pairs, preferably 400 to 1,500base pairs, and most preferably 800 to 1,500 base pairs, which arehighly homologous with the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding nucleotide sequences. On the other hand, thevector may be integrated into the genome of the host cell bynon-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. Examples of bacterial origins of replication are theorigins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184permitting replication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1permitting replication in Bacillus. Examples of origins of replicationfor use in a yeast host cell are the 2 micron origin of replication,ARS1, ARS4, the combination of ARS1 and CEN3, and the combination ofARS4 and CEN6. The origin of replication may be one having a mutationwhich makes its functioning temperature-sensitive in the host cell (see,e.g., Ehrlich, 1978, Proceedings of the National Academy of Sciences USA75: 1433).

More than one copy of a nucleotide sequence of the present invention maybe inserted into the host cell to increase production of the geneproduct. An increase in the copy number of the nucleotide sequence canbe obtained by integrating at least one additional copy of the sequenceinto the host cell genome or by including an amplifiable selectablemarker gene with the nucleotide sequence where cells containingamplified copies of the selectable marker gene, and thereby additionalcopies of the nucleotide sequence, can be selected for by cultivatingthe cells in the presence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see e.g. Sambrook et al., 1989, supra).

Recombinant Host Cell Comprising Nucleic Acid Construct

The present invention also relates to recombinant a host cell comprisingthe nucleic acid construct of the invention, which are advantageouslyused in the recombinant production of the polypeptides. A vectorcomprising a nucleotide sequence of the present invention is introducedinto a host cell so that the vector is maintained as a chromosomalintegrant or as a self-replicating extra-chromosomal vector as describedearlier.

The host cell may be a unicellular microorganism, e.g., a prokaryote ora non-unicellular microorganism, e.g., a eukaryote.

Useful unicellular cells are bacterial cells such as gram positivebacteria including, but not limited to, a Bacillus cell, e.g., Bacillusalkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacilluscirculans, Bacillus clausii, Bacillus coagulans, Bacillus lautus,Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis; or aStreptomyces cell, e.g., Streptomyces lividans or Streptomyces murinus,or gram negative bacteria such as E. coli and Pseudomonas sp. In apreferred embodiment, the bacterial host cell is a Bacillus lentus,Bacillus licheniformis, Bacillus stearothermophilus, or Bacillussubtilis cell. In another preferred embodiment, the Bacillus cell is analkalophilic Bacillus. (KKSC/SALK verify relevans for GH-61polypeptides)

The introduction of a vector into a bacterial host cell may, forinstance, be effected by protoplast transformation (see, e.g., Chang andCohen, 1979, Molecular General Genetics 168: 111–115), using competentcells (see, e.g., Young and Spizizin, 1961, Journal of Bacteriology 81:823–829, or Dubnau and Davidoff-Abelson, 1971, Journal of MolecularBiology 56: 209–221), electroporation (see, e.g., Shigekawa and Dower,1988, Biotechniques 6: 742–751), or conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5771–5278).

The host cell may be a eukaryote, such as a mammalian, insect, plant, orfungal cell.

In a preferred embodiment, the host cell is a fungal cell. “Fungi” asused herein includes the phyla Ascomycota, Basidiomycota,Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., In,Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CABInternational, University Press, Cambridge, UK) as well as the Oomycota(as cited in Hawksworth et al., 1995, supra, page 171) and allmitosporic fungi (Hawksworth et al., 1995, supra).

In a more preferred embodiment, the fungal host cell is a yeast cell.“Yeast” as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A., Passmore,S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium SeriesNo. 9,1980).

In an even more preferred embodiment, the yeast host cell is a Candida,Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, orYarrowia cell.

In a most preferred embodiment, the yeast host cell is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensisor Saccharomyces oviformis cell. In another most preferred embodiment,the yeast host cell is a Kluyveromyces lactis cell. In another mostpreferred embodiment, the yeast host cell is a Yarrowia lipolytica cell.

In another more preferred embodiment, the fungal host cell is afilamentous fungal cell. “Filamentous fungi” include all filamentousforms of the subdivision Eumycota and Oomycota (as defined by Hawksworthet al., 1995, supra). The filamentous fungi are characterized by amycelial wall composed of chitin, cellulose, glucan, chitosan, mannan,and other complex polysaccharides. Vegetative growth is by hyphalelongation and carbon catabolism is obligately aerobic. In contrast,vegetative growth by yeasts such as Saccharomyces cerevisiae is bybudding of a unicellular thallus and carbon catabolism may befermentative.

In an even more preferred embodiment, the filamentous fungal host cellis a cell of a species of, but not limited to, Acremonium, Aspergillus,Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium,Thielavia, Tolypocladium, or Trichoderma.

In a most preferred embodiment, the filamentous fungal host cell is anAspergillus awamori, Aspergillus foetidus, Aspergillus japonicus,Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae cell. Inanother most preferred embodiment, the filamentous fungal host cell is aFusarium bactridioides, Fusarium cerealis, Fusarium crookwellense,Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioides, or Fusarium venenatum cell. In an even mostpreferred embodiment, the filamentous fungal parent cell is a Fusariumvenenatum (Nirenberg sp. nov.) cell. In another most preferredembodiment, the filamentous fungal host cell is a Humicola insolens,Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila,Neurospora crassa, Penicillium purpurogenum, Thielavia terrestris,Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus host cells are described in EP 238 023 andYelton et al., 1984, Proceedings of the National Academy of Sciences USA81: 1470–1474. Suitable methods for transforming Fusarium species aredescribed by Malardier et al., 1989, Gene 78: 147–156 and WO 96/00787.Yeast may be transformed using the procedures described by Becker andGuarente, In Abelson, J. N. and Simon, M. I., editors, Guide to YeastGenetics and Molecular Biology, Methods in Enzymology, Volume 194, pp182–187, Academic Press, Inc., New York; Ito et al., 1983, Journal ofBacteriology 153: 163; and Hinnen et al., 1978, Proceedings of theNational Academy of Sciences USA 75: 1920.

Processes for Preparing Functional GH-61 Polypeptides

The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating astrain, which in its wild-type form is capable of producing thepolypeptide; and (b) recovering the polypeptide. Preferably, the strainis a fungus, more preferably of the genus Humicola, particularlyHumicola insolens or Coprinus, such as Coprinus cinereus or Thelaviasuch as Thelavia terrestris

The present invention also relates to methods for producing apolypeptide of the invention comprising (a) cultivating a host cellunder conditions conducive for production of the polypeptide; and (b)recovering the polypeptide.

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods known in the art. For example, the cell may becultivated by shake flask cultivation, small-scale or large-scalefermentation (including continuous, batch, fed-batch, or solid statefermentations) in laboratory or industrial fermentors performed in asuitable medium and under conditions allowing the polypeptide to beexpressed and/or isolated. The cultivation takes place in a suitablenutrient medium comprising carbon and nitrogen sources and inorganicsalts, using procedures known in the art. Suitable media are availablefrom commercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The polypeptide may be detected and/or identified using methods known inthe art and modifications thereof that are specific for the polypeptide.These detection methods may include use of specific antibodies,formation of a polypeptide—carbohydrate complex, or disappearance of anactivated carbohydrate substrate, sequencing and alignment, testing inmethods for preparing edible products etc. The resulting polypeptide maybe recovered by methods known in the art. For example, the polypeptidemay be recovered from the nutrient medium by conventional proceduresincluding, but not limited to, centrifugation, filtration, extraction,spray-drying, evaporation, or precipitation.

The polypeptides of the present invention may be purified by a varietyof procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.Protein Purification, J.-C. Janson and Lars Ryden, editors, VCHPublishers, New York, 1989).

Transgenic Plants

The present invention also relates to a transgenic plant, plant part, orplant cell which has been transformed with a nucleotide sequenceencoding a polypeptide of the present invention so as to express andproduce the polypeptide. In one embodiment the plant could be used ashost for production of polypeptide in recoverable quantities. Thepolypeptide may be recovered from the plant or plant part.Alternatively, the plant or plant part containing the recombinantpolypeptide may be used as such for as ingredients in a doughcomposition having improved quality. The transgenic plant can bedicotyledonous (a dicot) or monocotyledonous (a monocot). Examples ofmonocot plants are grasses, such as meadow grass (blue grass, Poa),forage grass such as festuca, lolium, temperate grass, such as Agrostis,and cereals, e.g., wheat, oats, rye, barley, rice, sorghum, and maize(corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers. Also specific plant tissues, such as chloroplast, apoplast,mitochondria, vacuole, peroxisomes, and cytoplasm are considered to be aplant part. Furthermore, any plant cell, whatever the tissue origin, isconsidered to be a plant part.

Also included within the scope of the present invention are the progenyof such plants, plant parts and plant cells.

The transgenic plant or plant cell expressing a polypeptide of thepresent invention may be constructed in accordance with methods known inthe art. Briefly, the plant or plant cell is constructed byincorporating one or more expression constructs encoding a polypeptideof the present invention into the plant host genome and propagating theresulting modified plant or plant cell into a transgenic plant or plantcell.

Conveniently, the expression construct is a nucleic acid construct whichcomprises a nucleotide sequence encoding a polypeptide of the presentinvention operably linked with appropriate regulatory sequences requiredfor expression of the nucleotide sequence in the plant or plant part ofchoice. Furthermore, the expression construct may comprise a selectablemarker useful for identifying host cells into which the expressionconstruct has been integrated and DNA sequences necessary forintroduction of the construct into the plant in question (the latterdepends on the DNA introduction method to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences, is determined, forexample, on the basis of when, where, and how the polypeptide is desiredto be expressed. For instance, the expression of the gene encoding apolypeptide of the present invention may be constitutive or inducible,or may be developmental, stage or tissue specific, and the gene productmay be targeted to a specific tissue or plant part such as seeds orleaves. Regulatory sequences are, for example, described by Tague etal., 1988, Plant Physiology 86: 506.

For constitutive expression, the 35S-CaMV promoter may be used (Francket al., 1980, Cell 21: 285–294). Organ-specific promoters may be, forexample, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards & Coruzzi, 1990, Ann. Rev. Genet. 24:275–303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863–878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant and Cell Physiology 39: 885–889), a Vicia faba promoterfrom the legumin B4 and the unknown seed protein gene from Vicia faba(Conrad et al., 1998, Journal of Plant Physiology 152: 708–711), apromoter from a seed oil body protein (Chen et al., 1998, Plant and CellPhysiology 39: 935–941), the storage protein napA promoter from Brassicanapus, or any other seed specific promoter known in the art, e.g., asdescribed in WO 91/14772. Furthermore, the promoter may be a leafspecific promoter such as the rbcs promoter from rice or tomato (Kyozukaet al., 1993, Plant Physiology 102: 991–1000, the chlorella virusadenine methyltransferase gene promoter (Mitra and Higgins, 1994, PlantMolecular Biology 26: 85–93), or the aldP gene promoter from rice(Kagaya et al., 1995, Molecular and General Genetics 248: 668–674), or awound inducible promoter such as the potato pin2 promoter (Xu et al.,1993, Plant Molecular Biology 22: 573–588).

A promoter enhancer element may also be used to achieve higherexpression of the polypeptide in the plant. For instance, the promoterenhancer element may be an intron which is placed between the promoterand the nucleotide sequence encoding an polypeptide of the presentinvention. For instance, Xu et al., 1993, supra disclose the use of thefirst intron of the rice actin 1 gene to enhance expression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Presently, Agrobacterium tumefaciens-mediated gene transfer is themethod of choice for generating transgenic dicots (for a review, seeHooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15–38).However it can also be used for transforming monocots, although othertransformation methods are generally preferred for these plants.Presently, the method of choice for generating transgenic monocots isparticle bombardment (microscopic gold or tungsten particles coated withthe transforming DNA) of embryonic calli or developing embryos(Christou, 1992, Plant Journal 2: 275–281; Shimamoto, 1994, CurrentOpinion Biotechnology 5: 158–162; Vasil et al., 1992, Bio/Technology 10:667–674). An alternative method for transformation of monocots is basedon protoplast transformation as described by Omirulleh et al., 1993,Plant Molecular Biology 21: 415–428.

Following transformation, the transformants having incorporated thereinthe expression construct are selected and regenerated into whole plantsaccording to methods well-known in the art.

The present invention also relates to methods for producing anpolypeptide of the present invention comprising (a) cultivating atransgenic plant or a plant cell comprising a nucleotide sequenceencoding an polypeptide of the present invention under conditionsconducive for production of the polypeptide; and (b) recovering thepolypeptide.

Compositions Comprising Functional GH-61 Polypeptides

In a still further aspect, the present invention relates to compositionscomprising a polypeptide of the present invention and their preparation,in particular compositions wherein the polypeptide of the invention isthe major component of the composition, e.g., a mono-componentcomposition.

The composition may further comprise one or more enzymes, in particularcarbohydrases such as amylase, glucanase, polypeptide, galactanase,mannanase etc, The enzymes may also include enzymes such asaminopeptidase, carboxypeptidase, catalase, chitinase, cutinase,cyclodextrin glycosyltransferase, deoxyribonuclease, esterase,alpha-galactosidase, beta-galactosidase, glucoamylase,alpha-glucosidase, beta-glucosidase, haloperoxidase, invertase, laccase,lipase, mannosidase, oxidase, pectinolytic enzymes, peptidoglutaminase,peroxidase, phytase, polyphenoloxidase, proteolytic enzyme,ribonuclease, transglutaminase, or polypeptide. In a particularembodiment the amylase is a maltogenic amylase, especially a maltogenicexo-amylase such as maltogenic exo-alpha-amylase or maltogenicexo-beta-amylase.

The compositions may be prepared in accordance with methods known in theart and may have any physical appearance such as liquid, paste or solid.For instance, the polypeptide composition may be formulated usingmethods known to the art of formulating enzymes and/or pharmaceuticalproducts, e.g. into coated or uncoated granules or micro-granules. Thepolypeptide to be included in the composition may be stabilized inaccordance with methods known in the art e.g. by stabilizing thepolypeptide in the composition by adding and antioxidant or reducingagent to limit oxidation or the polypeptide of it may be stabilized byadding polymers such as PVP, PVA, PEG or other suitable polymers knownto be beneficial to the stability of polypeptides in solid or liquidcompositions. When formulating GH-61 polypeptides of the invention as agranulate or agglomerated powder the particles particularly have anarrow particle size distribution with more than 95% (by weight) of theparticles in the range from 25 to 500 μm. Granulates and agglomeratedpowders may be prepared by conventional methods, e.g. by spraying theamylase onto a carrier in a fluid-bed granulator. The carrier mayconsist of particulate cores having a suitable particle size. Thecarrier may be soluble or insoluble, e.g. a salt (such as NaCl or sodiumsulfate), a sugar (such as sucrose or lactose), a sugar alcohol (such assorbitol), starch, rice, corn grits, or soy. Hence the invention alsoprovides a granule comprising a GH-61polypeptide

In a particular embodiment the composition is a dough composition or adough improving additive comprising a GH-61 polypeptide of theinvention.

The dough may comprise basis ingredients such as meal, flour or starchsuch as wheat meal, wheat flour, corn flour, corn starch, rye meal, ryeflour, oat flour, oat meal, sorghum meal, sorghum flour, rice flour,potato meal, potato flour or potato starch.

The dough may also comprise other conventional dough ingredients, e.g.proteins, such as milk powder and gluten; eggs (either whole eggs, eggyolks or egg whites); oxidants such as ascorbic acid, potassium bromate,potassium iodate, azodicarbonamide (ADA) or ammonium persulfate; aminoacids such as L-cysteine and/or glutamate; sugars; salts such as sodiumchloride, calcium acetate, sodium sulfate or calcium sulfate.

The dough may still further comprise fat (triglyceride) such asgranulated fat or shortening.

The dough or dough improving additive may still further comprise anemulsifier such as mono- or diglycerides, diacetyl tartaric acid estersof mono- or diglycerides, sugar esters of fatty acids, polyglycerolesters of fatty acids, lactic acid esters of monoglycerides, acetic acidesters of monoglycerides, polyoxyethylene stearates, or lysolecithin.

The dough or dough improving additive may still further comprise aleavening agent such as yeast, usually Saccharomyces cerevisiae (baker'syeast) and/or chemical leaving agents such as bicarbonate compounds usedin baking powder.

The dough or dough improving additive may further comprise additionalenzymes. Such enzymes includes a lipolytic enzyme, particularlyphospholipase, galactoilipase and/or triacyl glycerol lipase activity,e.g. as described in WO 9953769, WO 0032758, WO 0200852 or WO2002066622. Other enzymes may be a amylases, cyclodextringlucanotransferase, protease or peptidase, in particular anexopeptidase, transglutaminase, lipase, cellulase, hemicellulase,glycosyltransferase, branching enzyme (1,4-□-glucan branching enzyme) oroxidoreductase. The additional enzyme may be of mammalian, plant ormicrobial (bacterial, yeast or fungal) origin. The amylase may be from afungus, bacterium or plant. It may be a maltogenic alpha-amylase (EC3.2.1.133), e.g. from B. stearothermophilus, an alpha-amylase, e.g. fromBacillus, particularly B. licheniformis or B. amyloliquefaciens, abeta-amylase, e.g. from plant (e.g. soy bean) or from microbial sources(e.g. Bacillus), a glucoamylase, e.g. from A. niger, or a fungalalpha-amylase, e.g. from A. oryzae. The hemicellulase may be apentosanase, e.g. a xylanase which may be of microbial origin, e.g.derived from a bacterium or fungus, such as a strain of Aspergillus, inparticular of A. aculeatus, A. niger, A. awamori, or A. tubigensis, froma strain of Trichoderma, e.g. T. reesei, or from a strain of Humicola,e.g. H. insolens. The protease may be from Bacillus, e.g. B.amyloliquefaciens. The oxidoreductase may be a glucose oxidase, a hexoseoxidase, a lipoxidase, a peroxidase, or a laccase.

The dough may still further appear as fresh, frozen or par-baked dough.It may also be laminated dough.

The amount of GH-61 polypeptide in the composition, particularly thedough, should amount to between 0.5–100 mg polypeptide per kg dry matterin the dough, in particular 0.5–50 mg polypeptide per kg dry matter, inparticular 1–25 mg polypeptide per kg dry matter, in particular 1–15 mgpolypeptide per kg dry matter in the dough, in particular 2–10 mg/kg.

Considering the findings that the GH-61 polypeptides of the inventionhave a significant effect in dough compositions it is presentlycontemplated that as soling in laundry most often also containsfoodstuffs it is will also have an effect in removing such soilings fromtextile in a washing process. Hence in a further embodiment, thecomposition of the invention is a detergent composition which, inaddition to the GH-61 polypeptide of the invention, comprises asurfactant and optionally compounds selected from the group consistingof builders such as zeolites, bleaching agents such as percarbonate,bleach enhancers such as TAED or NOBS, suds suppressors, fragrants, etc.

In a further embodiment, the composition of the invention is a cerealcontaining feed composition which, in addition to the polypeptide,comprises a cereal or grain product.

In a further embodiment, the composition of the invention is fermentablecomposition, which in addition to the polypeptide, comprises one or morenutrients for a microorganism.

In a further embodiment, the composition of the invention is a pulpingcomposition, which in addition to the polypeptide, comprises pulp.

Applications of Functional GH-61 Polypeptides

The first aspect of the invention relates to finding that isolated GH-61polypeptides had significant anti-staling effect when used (in effectiveamounts for providing an anti-staling effect) for preparing edibleproducts and thus the invention provides use of an anti-staling GH-61polypeptide for preparing an edible product. This use may in particularinvolve a method of preparing an edible product comprising heating adough composition comprising an effective amount of anti-staling GH-61polypeptide; in particular the method comprises leavening and heating adough composition. An effective amount of GH-61 polypeptide is theminimum amount required to provide a measurable anti-staling effect inan edible product.

A contemplated embodiment of GH-61 polypeptides of the invention is theuse of effective amounts of GH-61 polypeptides for preparation of acereal containing feed composition comprising mixing a feed mixture witha polypeptide of the invention.

In yet another contemplated embodiment, effective amounts of the GH-61polypeptide of the invention may be applied in a process hydrolysis ofagricultural wastes for production of alcohol fuels.

In yet another contemplated embodiment effective amounts of the GH-61polypeptide of the invention may be applied in a brewing process whereinthe presence of a polypeptide may improve filterability of the wort.

In yet another contemplated embodiment, effective amounts of the GH-61polypeptide of the invention may be applied in a process for preparationof fruit or vegetable juices, wherein presence of a polypeptide mayimprove filtration and increase yields.

In yet another contemplated embodiment effective amounts of the GH-61polypeptide of the invention may be applied in a process for treatmentof lignolosic materials and pulp with a polypeptide.

Detergent Disclosure and Examples

The polypeptide of the invention may be added to and thus become acomponent of a detergent composition.

The detergent composition of the invention may for example be formulatedas a hand or machine laundry detergent composition including a laundryadditive composition suitable for pre-treatment of stained fabrics and arinse added fabric softener composition, or be formulated as a detergentcomposition for use in general household hard surface cleaningoperations, or be formulated for hand or machine dishwashing operations.

In a specific aspect, the invention provides a detergent additivecomprising the enzyme of the invention. The detergent additive as wellas the detergent composition may comprise one or more other enzymes suchas a protease, a lipase, a cutinase, an amylase, a carbohydrase, acellulase, a pectinase, a mannanase, an arabinase, a galactanase, axylanase, an oxidase, e.g., a laccase, and/or a peroxidase.

In general the properties of the chosen enzyme(s) should be compatiblewith the selected detergent, (i.e. pH-optimum, compatibility with otherenzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) shouldbe present in effective amounts.

Proteases: Suitable proteases include those of animal, vegetable ormicrobial origin. Microbial origin is preferred. Chemically modified orprotein engineered mutants are included. The protease may be a serineprotease or a metallo protease, preferably an alkaline microbialprotease or a trypsin-like protease. Examples of alkaline proteases aresubtilisins, especially those derived from Bacillus, e.g., subtilisinNovo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 andsubtilisin 168 (described in WO 89/06279). Examples of trypsin-likeproteases are trypsin (e.g. of porcine or bovine origin) and theFusarium protease described in WO 89/06270 and WO 94/25583.

Examples of useful proteases are the variants described in WO 92/19729,WO 98/20115, WO 98/20116, and WO 98/34946, especially the variants withsubstitutions in one or more of the following positions: 27, 36, 57, 76,87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235 and274.

Preferred commercially available protease enzymes include Alcalase®,Savinase®, Primase®, Duralase®, Esperase®, and Kannase® (Novozymes A/S),Maxatase®, Maxacal®, Maxapem®, Properase®, Purafect®, Purafect OxP®,FN2®), and FN3® (Genencor International Inc.).

Lipases: Suitable lipases include those of bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Examplesof useful lipases include lipases from Humicola (synonym Thermomyces),e.g. from H. lanuginosa (T. lanuginosus) as described in EP 258 068 andEP 305 216 or from H. insolens as described in WO 96/13580, aPseudomonaslipase, e.g. from P. alcaligenes or P. pseudoalcaligenes (EP218 272), P. cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P.fluorescens, Pseudomonas sp. strain SD 705 (WO 95/06720 and WO96/27002), P. wisconsinensis (WO 96/12012), a Bacillus lipase, e.g. fromB. subtilis (Dartois et al. (1993), Biochemica et Biophysica Acta, 1131,253–360), B. stearothermophilus (JP 64/744992) or B. pumilus (WO91/16422).

Other examples are lipase variants such as those described in WO92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292,WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO97/07202.

Preferred commercially available lipase enzymes include LipolaseTM andLipolase UltraTM (Novozymes A/S).

Amylases: Suitable amylases (alpha and/or beta) include those ofbacterial or fungal origin. Chemically modified or protein engineeredmutants are included. Amylases include, for example, □-amylases obtainedfrom Bacillus, e.g. a special strain of B. licheniformis, described inmore detail in GB 1,296,839.

Examples of useful amylases are the variants described in WO 94/02597,WO 94/18314, WO 96/23873, and WO 97/43424, especially the variants withsubstitutions in one or more of the following positions: 15, 23, 105,106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243,264, 304, 305, 391, 408, and 444.

Commercially available amylases are DuraMyl™, TermaMyl™, FungaMyl™ andBAN™ (Novozymes A/S), Rapidase™ and Purastar™ (from GenencorInternational Inc.).

Cellulases: Suitable cellulases include those of bacterial or fungalorigin. Chemically modified or protein engineered mutants are included.Suitable cellulases include cellulases from the genera Bacillus,Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g. the fungalcellulases produced from Humicola insolens, Myceliophthora thermophilaand Fusarium oxysporum disclosed in U.S. Pat. Nos. 4,435,307, 5,648,263,5,691,178, 5,776,757 and WO 89/09259.

Especially suitable cellulases are the alkaline or neutral cellulaseshaving colour care benefits. Examples of such cellulases are cellulasesdescribed in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO98/08940. Other examples are cellulase variants such as those describedin WO 94/07998, EP 0 531 315, U.S. Pat. Nos. 5,457,046, 5,686,593,5,763,254, WO 95/24471, WO 98/12307 and PCT/DK98/00299.

Commercially available cellulases include Celluzyme®, and Carezyme®(Novozymes), Clazinase®, and Puradax HA® (Genencor International Inc.),and KAC-500(B)® (Kao Corporation).

Peroxidases/Oxidases: Suitable peroxidases/oxidases include those ofplant, bacterial or fungal origin. Chemically modified or proteinengineered mutants are included. Examples of useful peroxidases includeperoxidases from Coprinus, e.g. from C. cinereus, and variants thereofas those described in WO 93/24618, WO 95/10602, and WO 98/15257.

Commercially available peroxidases include Guardzyme® (Novozymes A/S).

The detergent enzyme(s) may be included in a detergent composition byadding separate additives containing one or more enzymes, or by adding acombined additive comprising all of these enzymes. A detergent additiveof the invention, i.e. a separate additive or a combined additive, canbe formulated e.g. as a granulate, a liquid, a slurry, etc. Preferreddetergent additive formulations are granulates, in particularnon-dusting granulates, liquids, in particular stabilized liquids, orslurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethyleneglycol, PEG) with mean molar weights of1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238,216.

The detergent composition of the invention may be in any convenientform, e.g. a bar, a tablet, a powder, a granule, a paste or a liquid. Aliquid detergent may be aqueous, typically containing up to 70% waterand 0–30% organic solvent, or non-aqueous.

The detergent composition comprises one or more surfactants, which maybe non-ionic including semi-polar and/or anionic and/or cationic and/orzwitterionic. The surfactants are typically present at a level of from0.1% to 60% by weight.

When included therein the detergent will usually contain from about 1%to about 40% of an anionic surfactant such as linearalkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fattyalcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate,alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid orsoap.

When included therein the detergent will usually contain from about 0.2%to about 40% of a non-ionic surfactant such as alcohol ethoxylate,nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide,ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide,polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives ofglucosamine (“glucamides”).

The detergent may contain 0–65% of a detergent builder or complexingagent such as zeolite, diphosphate, triphosphate, phosphonate,carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraaceticacid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinicacid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).

The detergent may comprise one or more polymers. Examples arecarboxymethylcellulose, poly(vinylpyrrolidone), poly (ethylene glycol),poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole),polycarboxylates such as polyacrylates, maleic/acrylic acid copolymersand lauryl methacrylate/acrylic acid copolymers.

The detergent may contain a bleaching system which may comprise a H2O2source such as perborate or percarbonate which may be combined with aperacid-forming bleach activator such as tetraacetylethylenediamine ornonanoyloxybenzenesulfonate. Alternatively, the bleaching system maycomprise peroxyacids of e.g. the amide, imide, or sulfone type.

The enzyme(s) of the detergent composition of the invention may bestabilized using conventional stabilizing agents, e.g., a polyol such aspropylene glycol or glycerol, a sugar or sugar alcohol, lactic acid,boric acid, or a boric acid derivative, e.g., an aromatic borate ester,or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid,and the composition may be formulated as described in e.g. WO 92/19709and WO 92/19708.

The detergent may also contain other conventional detergent ingredientssuch as e.g. fabric conditioners including clays, foam boosters, sudssuppressors, anti-corrosion agents, soil-suspending agents, anti-soilredeposition agents, dyes, bactericides, optical brighteners,hydro-tropes, tarnish inhibitors, or perfumes.

It is at present contemplated that in the detergent compositions anyenzyme, in particular the enzyme of the invention, may be added in anamount corresponding to 0.01–100 mg of enzyme protein per liter of washliquor, preferably 0.05–5 mg of enzyme protein per liter of wash liquor,in particular 0.1–1 mg of enzyme protein per liter of wash liquor.

The enzyme of the invention may additionally be incorporated in thedetergent formulations disclosed in WO 97/07202 which is herebyincorporated as reference.

EXAMPLES Example 1 Isolation of DNA Including GH-61 Polypeptides fromThe lavia terrestris

Mycelium of a T. terrestris is grown on MEX-1 to induce proteins thatrespond to complex cellulosic medias. After 4 days at 37° C., themycelium is harvested by filtration though Whatman 1MM filter paper.Mycelium is frozen in liquid nitrogen and stored until further use. RNAisolation is performed according to the protocol of Chomczyniski andSacchi, 1987 (Analytical Biochemistry 162: 156–159). The Poly(A) QuikmRNA isolation kit is used to purify polyA enriched RNA for cDNAproduction (Stratagene USA). Production of a cDNA library is achievedaccording to the SMART cDNA library construction kit (Clontech USA).Sfil restricted double strand cDNAs are cloned into Lambda TriplExvector and the plasmid containing colonies is recovered by mass excisionaccording to the SMART protocol.

Individual plasmids containing cDNAs are prepared using existing 96 wellsilica based plasmid preparation systems such as Qiagen Qia-turbo 96,Qiagen corp. USA). Once plasmid template is prepared, all plasmids aresequenced with the vector forward primer using conventional sequencingmethods and equipment.

Identification of GH61 Expressed Sequence Tags (ESTs) are achieved bysearching the entire non redundant protein database (for example, SWALL)with a program such as BlastP (Altschul, S. F., Gish, W., Miller, W.,Myers, E. W. & Lipman, D. J. (1990) “Basic local alignment search tool.”J. Mol. Biol. 215:403–410. Gish, W. & States, D. J. (1993)“Identification of protein coding regions by database similaritysearch.” Nature Genet. 3:266–272. Madden, T. L., Tatusov, R. L. & Zhang,J. (1996) “Applications of network BLAST server” Meth. Enzymol.266:131–141. Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J.,Zhang, Z., Miller, W. & Lipman, D. J. (1997) “Gapped BLAST andPSI-BLAST: a new generation of protein database search programs.”Nucleic Acids Res. 25:3389–3402. Zhang, J. & Madden, T. L. (1997)“PowerBLAST: A new network BLAST application for interactive orautomated sequence analysis and annotation.” Genome Res. 7:649–656.

Sequences with similarities to existing GH-61 family sequences areidentified based on a blast probability score matrix.

A clone containing SEQ ID NO: 1 was selected for further analysis

Example 2 Isolation of DNA Encoding GH-61 Polypeptides from Coprinuscinereus

The procedure of example 1 was repeated for a strain of Humicolainsolens, A clone containing SEQ ID NO: 3 was selected for furtheranalysis.

Example 3 Isolation Of DNA Encoding GH-61 Polypeptides from Humicolainsolens

The procedure of example 1 was repeated for a strain of Coprinuscinereus for example Coprinius cinerus (CBS394.65) obtainable from theCBS. A clone containing SEQ ID NO: 5 was selected for further analysis.

Example 4 Construction of an Aspergillus Expression Vector for GH61 DNASequences

SEQ ID NO: 3 was amplified in the following manner: 1 microliter of cDNA(approximately 10 nanograms of DNA) was used as template in a PCRreaction with the two primers A and B.

(SEQ ID NO:7) Primer A: 5′-GCGGAATTCATCATGAGGCCCTTCTCCCTC-3′ (SEQ IDNO:8) Primer B: 5′-ATTTGCGGCCGCTTCCCGTCATCCTCTAAGGC-3′SEQ ID NO: 1 and 5 can be amplified in a similar manner using theprimers:For SEQ. ID NO: 1:

(SEQ ID NO:9) Primer A: 5′-GCGGAATTCATGAAGCTCACCACCTCGGT-3′ (SEQ IDNO:10) Primer B: 5′-ATTTGCGGCCGCGCAGCCAACCAACCTGGAAT-3′For SEQ ID NO: 5:

(SEQ ID NO:11) Primer A: 5′-GCGGAATTCACAATGAAGGTCTTCGCATAC-3′ (SEQ IDNO:12) primer B: 5′-ATTTGCGGCCGCACGATGCGATGAGCATTTAT-3′

5 pmoles of each primer was used in a 50 microliter reaction volume. TheQiagen ProofStart high fidelity DNA polymerase and buffer were usedaccording to the manufacturer's instructions (Qiagen, USA). Briefly, thereaction was placed in a thermal cycler (MJ Research, Dyad, USA) andcycled under the following reaction conditions: An initial denaturationof 5 minutes at 95 degrees Celsius, 25 cycles of the following: 94degrees-30 seconds, 55 degrees-30 seconds, 72 degrees 2 minutes. A finalextension temperature of 72 degrees for 10 minutes was then used.Aliquots of the PCR reaction were separated on a 1% agarose gel. Onedistinct band was seen: The size of this band (1.1 kb) corresponded wellwith the predicted size of the open reading frame.

The fragment was digested with EcoRI and NotI which cut in the overhangsintroduced by the PCR primers. The digested fragments were isolated andcloned into pMStr54, an Aspergillus expression plasmid based on theplasmid pCaHj527 (see the examples of international patent applicationWO 00/70064) constructed as described in example 7 of WO 02/12472.Plasmid DNA was isolated from colonies of the cloning experiment. Thecolonies were sequenced with vector primers PNA21 (5′-GTT TCC MC TCA ATTTAC CTC-3′) and MHas5NotI (5′-TTG CCC TCA TCC CCA TCC TTT-3′) whichprime in opposite directions into the plasmid insert. It was determinedthat no errors were introduced in any of the insert sequences as aresult of PCR.

Example 5 Expression of SEQ ID NOS: 1, 3 and 5 in Aspergillus:

SEQ ID NOS: 1, 3 and 5 were transformed into Aspergillus oryzae strainJAL355 (disclosed in international patent application WO 01/98484A1).Transformants of SEQ ID NOS: 1,3 and 5 were re-isolated twice underselective and noninducing conditions on Cove minimal plates with 1Msucrose as a carbon source and 10 mM nitrate. (See recipe of themanufacturer) To test expression of SEQ ID NOS: 1,3 and 5, transformantswere grown for 3 days and 4 days at 30 degrees celsius in tubes with 10ml YPM (2% peptone, 1% yeast extract, 2% maltose). Supernatants were runon NuPage 10% Bis-Tris SDS gels (Invitrogen) as recommended by themanufacturer. All Aspergillus isolates grew well even when induced forthe expression of SEQ ID NOS: 1, 3 and 5.

Example 6 Purification of SEQ ID NO: 2 from Expression of SEQ ID NOS: 1in Aspergillus:

A culture supernatant from a fermentation of the Aspergillus oryzaestrain expressing SEQ ID NO: 1 was filtered through a 0.22 μm filter toremove the mycelia. 350 ml of the filtered supernatant was diluted to1450 ml with water and pH adjusted to 7.5 resulting in a conductivity of1.8 mS/cm. This solution was loaded onto a 50 ml Q-Sepharose anionexchange column equilibrated with 25 mM Tris pH 7.5. The column waswashed with about 15 column volumes of 25 mM Tris pH 7.5 and boundproteins were eluted with a NaCl gradient increasing linearly from 0 to0.5 M over 20 column volumes. From SDS-PAGE it was seen that apolypeptide with a molecular weight of about 29 kDa was eluted duringthe wash with 25 mM Tris, pH 7.5. Fractions containing the 29 kDapolypeptide were pooled and concentrated on an Amicon ultrafiltrationdevice with a 6 kDa cut off filter (Dow, GR 81PP). The concentrated poolwas at least 95% pure as estimated from SDS-PAGE and N-terminalsequencing of the polypeptide gave the sequence corresponding to SEQ IDNO: 2 (HYTFPQTDINGQLSGE).

Example 7 Purification of SEQ ID NO: 4 from Expression of SEQ ID NOS: 3in Aspergillus:

A culture supernatant from a fermentation of the Aspergillus oryzaestrain expressing SEQ ID NO: 3 was filtered through a 0.22 μm filter toremove the mycelia. 500 ml of the filtered supernatant wasultrafiltrated in a Filtron device with a 10 kDa cut off membrane. pHwas adjusted to 9.5 and the filtrate loaded onto a 50 ml Q-Sepharoseanion exchange column equilibrated with 25 mM glycine pH 9.5. The columnwas washed with about two column volumes of 25 mM glycine pH 9.5 andbound proteins were eluted with a NaCl gradient increasing linearly from0 to 0.5 M over 20 column volumes. From SDS-PAGE it was seen that apolypeptide with a molecular weight of about 32 kDa was eluted duringthe wash with 25 mM glycine pH 9.5. Fractions containing the 32 kDapolypeptide were pooled and concentrated on an Amicon ultrafiltrationdevice with a 20 kDa cut off filter (DDS, GR 61PP). Purity of theconcentrated pool was around 85% as estimated from SDS-PAGE andconcentration around 2.4 mg/ml estimated from absorbance at 280 nm andtheoretical extinction coefficient.

Example 8 Testing Activity of SEQ ID NOS: 2, 4 and 6

An oat AZCL-xylan, a birchwood AZCL-xylan and a wheat AZCL arabinoxylansubstrate suspension were prepared by suspending 2 milligram permilliliter of the substrate in a Nap buffer, pH 7 containing 0.0225% w/wBrij.

Activity of the purified SEQ ID NOS 2, 4 and 6 were tested by mixing 500microliter substrate suspension with 100 microliter of a solutioncontaining isolated SEQ ID NOS 2, 4 or 6. This mixture was incubated at37° C., wherein after undigested substrate was sedimentated bycentrifugation and digested substrate was evaluated by measuringabsorbance of the supernatant at 590 nanometers. A blank valuedetermined by replacing for one sample the enzyme solution with a bufferwas subtracted the absorbance measurements.

Results:

Absorbance for Absorbance for GH-61 Absorbance for Birchwood AZCL- WheatAZCL- polypeptide Oat AZCL-xylan xylan arabinoxylan SEQ ID NO: 2 0.01720.1077 0.1296 SEQ ID NO: 4 0.0798 0.4017 0.8128 SEQ ID NO: 6 0.05360.2064 0.6101

These results indicate that the GH-61 polypeptides have at least a minoractivity against these substrates.

Example 9 Testing Effect of SEQ ID NOS: 2 and 4 on Quality of BakedBread

Bread was baked according to the Sponge & Dough method from 2 kg offlour. Ca propionate was added to the recipe. The Sponge & Dough methodis a recognized standard method well known to the skilled person, seefor example Bread & Bread Making; Mauri Integrated Ingredient, 10thEdition, November 1995, chapter 3.3.

Enzymes were dosed according to the table below:

1 2 3 4 5 6 7 8 Novamyl 400 400 400 400 400 400 400 400 MANU/kgShearzyme 200 FXU/kg H. insolens  4  8 SEQ ID NO: 4 mg/kg T. terrestris 1  2  5 SEQ ID NO: 2 mg/kgBread was packed in plastic bags and stored at room temperature untilanalysis. Texture and NMR were measured on day 7, 14 and 21 and a smallsensory evaluation was performed on day 21.Texture MeasurementsFirmness and elasticity data are shown in table 1 & 2.

TABLE 1 Change in Firmness during storage Day Firmness (g) 7 14 21 400MANU/kg Novamyl 379 531 784 400 MANU/kg Novamyl + 4 mg/kg SEQ ID NO: 4455 630 866 400 MANU/kg Novamyl + 8 mg/kg SEQ ID NO: 4 494 691 778 400MANU/kg Novamyl + 1 mg/kg SEQ ID NO: 2 442 642 871 400 MANU/kg Novamyl +2 mg/kg SEQ ID NO: 2 405 539 618 400 MANU/kg Novamyl + 5 mg/kg SEQ IDNO: 2 434 530 788 400 MANU/kg Novamyl 461 580 671 400 MANU/kg Novamyl +200 FXU/kg Shearzyme 518 773 986

TABLE 2 Change in Elasticity during storage Day Elasticity % (g/g) 7 1421 400 MANU/kg Novamyl 52.8 49.9 47.9 400 MANU/kg Novamyl + 4 mg/kg SEQID NO: 4 51.3 47.5 46.1 400 MANU/kg Novamyl + 8 mg/kg SEQ ID NO: 4 52.247.8 48.6 400 MANU/kg Novamyl + 1 mg/kg SEQ ID NO: 2 52.7 48.0 46.9 400MANU/kg Novamyl + 2 mg/kg SEQ ID NO: 2 51.9 48.8 48.5 400 MANU/kgNovamyl + 5 mg/kg SEQ ID NO: 2 51.9 49.5 47.0 400 MANU/kg Novamyl 52.649.2 48.8 400 MANU/kg Novamyl + 200 FXU/kg Shearzyme 51.1 46.5 44.8

The GH 61 polypeptides show promising results compared to the leadcombination=Novamyl+Shearzyme. Especially SEQ ID NO:2 in combinationwith Novamyl shows anti-staling effect on softness and elasticitycomparable to or better than Novamyl alone.

Water Characteristics

Amount and mobility of water was measured by NMR. The amount andmobility of the free water, believed to correlate with the moist feelingof bread, is shown in table 3.

TABLE 3 Change in water mobility with time (NMR) Day Mobility of freewater (μs) 7 14 21 400 MANU/kg Novamyl 7398 6801 6015 400 MANU/kgNovamyl + 4 mg/kg SEQ ID NO: 4 7333 6977 5994 400 MANU/kg Novamyl + 8mg/kg SEQ ID NO: 4 7243 6721 6302 400 MANU/kg Novamyl + 1 mg/kg SEQ IDNO: 2 7116 6617 6035 400 MANU/kg Novamyl + 2 mg/kg SEQ ID NO: 2 75527047 6221 400 MANU/kg Novamyl + 5 mg/kg SEQ ID NO: 2 7360 6942 6077 400MANU/kg Novamyl 7582 7067 6227 400 MANU/kg Novamyl + 200 FXU/kgShearzyme 7359 6686 6039

The two Novamyl references show very different results in themeasurement of water mobility by NMR. The “best” of the Novamylreferences show comparable effect to SEQ ID NO:2 in combination withNovamyl, however the second Novamyl reference show significantlyinferior results to these.

Sensory Evaluation

A sensory evaluation was made by a panel of people skilled in the art ofbaking bread. The bread with the GH61 polypeptide of SEQ ID NO:2 incombinations with Novamyl was picked as the very best regardingperception of moistness and softness. This sensory evaluation correlateswith migration of free water (measured by NMR), which can be related tomoistness.

Conclusion

The GH-61 polypeptides—especially SEQ ID NO:2—show significantanti-staling effects and improves the quality of bread prepared usingthe polypeptides. In particular fresh-keeping of bread, in particularsoftness, elasticity, perception of moistness and moisture retentioncapacity is improved.

Also the combination of GH-61 polypeptides with for example Novamyl,which is a maltogenic exo-amylase show improvements effect on softnessof bread, measured by texture analysis and at the same time elasticitywas more or less maintained compared to Novamyl alone.

1. An isolated glycoside hydrolase 61 (GH-61) polypeptide which isselected from the group consisting of: (a) a polypeptide that has anamino acid sequence which has at least 95% identity to: amino acids1-216 of SEQ ID NO: 2, amino acids 1-304 of SEQ ID NO: 4, or amino acids1-204 of SEQ ID NO: 6; (b) a polypeptide which is encoded by anucleotide sequence which hybridizes under high stringency conditionswith any of the following polynucleotide probes: (i) the complementarystrand of nucleotides 52–699 of SEQ ID NO: 1, 46–957 of SEQ ID NO: 3, or58–660 of SEQ ID NO: 5, (ii) the complementary strand of nucleotides 46to 857 of SEQ ID NO: 3, (iii) the complementary strand of nucleotides52–300 of SEQ ID NO: 1, 46–501 of SEQ ID NO: 3, or 58–300 of SEQ ID NO:5, and (iv) the complementary strand of nucleotides 301–699 of SEQ IDNO: 1, 502–957 of SEQ ID NO: 3, or 301–660 of SEQ ID NO:
 5. 2. Thepolypeptide of claim 1, which has an amino acid sequence which has atleast 95% identity with amino acids 1–216 of SEQ ID NO:
 2. 3. Thepolypeptide of claim 1, which has an amino acid sequence which comprisesamino acids 1–216 of SEQ ID NO:
 2. 4. The polypeptide of claim 1, whichhas an amino acid sequence which has at least 95% identity with aminoacids 1–304 of SEQ ID NO:
 4. 5. The polypeptide of claim 1, which has anamino acid sequence which comprises amino acids 1–304 of SEQ ID NO: 4.6. The polypeptide of claim 1, which has an amino acid sequence whichhas at least 95% identity with amino acids 1–201 of SEQ ID NO:
 6. 7. Thepolypeptide of claim 1, which has an amino acid sequence which comprisesamino acids 1–201 of SEQ ID NO:
 6. 8. The polypeptide of claim 1,wherein the polypeptide differs from amino acids of the maturepolypeptide of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 by at the mostten amino acids.
 9. The polypeptide of claim 1, wherein the polypeptideconsists of the amino acid sequence of the mature polypeptide of SEQ IDNO: 2, SEQ ID NO: 4 or SEQ ID NO:
 6. 10. The polypeptide of claim 1,comprising an amino acid sequence having H at position 1, A or P atposition 59, G at position 60, G at position 75, P or A at position 76,W or F at position 100, F or T at position 101, K or C at position 102,I or V or L at position 103, L or I or V or M at position 130, P atposition 131, G at position 137, Y at position 139, L or V or I or M atposition 140, L or V or I or M at position 141, R at position 142, E orQ at positions 143–144, L or V or I at position 148, H or N at position149, C at position 163 and P and G and P at positions 209–211respectively.
 11. The polypeptide of claim 1, which is encoded by anucleotide sequence which hybridizes under high stringency conditionswith any of the following polynucleotide probes: (a) the complementarystrand of nucleotides 52–699 of SEQ ID NO: 1, 46–957 of SEQ ID NO: 3, or58–660 of SEQ ID NO: 5, (b) the complementary strand of nucleotides 48to 857 of SEQ ID NO: 3, (c) the complementary strand of nucleotides52–300 of SEQ ID NO: 1, 46–501 of SEQ ID NO: 3, or 58–300 of SEQ ID NO:5, and (d) the complementary strand of nucleotides 301–699 of SEQ ID NO:1, 502–957 of SEQ ID NO: 3, or 301–660 of SEQ ID NO:
 5. 12. A method forpreparing an edible product, comprising: (a) adding a glycosidehydrolase 61 (GH-61) polypeptide of claim 1 to a dough in an amounteffective to retard the staling of the edible product prepared from thedough composition; and (b) heating the dough composition.
 13. The methodof claim 12, further comprising leavening the dough composition beforeheating.
 14. The method of claim 12, wherein the heating comprisesbaking the dough composition.
 15. The method of claim 12, wherein theheating comprises steaming the dough composition.
 16. The method ofclaim 12, wherein the edible product is a bread.
 17. The method of claim12, further comprising adding a maltogenic amylase to the doughcomposition.
 18. A dough composition, comprising a glycoside hydrolase61 (GH-61) polypeptide of claim 1 and at least one ingredient selectedfrom the group consisting of meal, flour and starch.
 19. The doughcomposition of claim 18, wherein the GH-61 polypeptide is in the form ofa granule.
 20. The dough composition of claim 18, wherein dough isfresh, frozen, par-baked or laminated dough.
 21. The dough compositionof claim 18, wherein the GH-61 polypeptide is added in an amount of0.5–100 mg GH-61 polypeptide per kg dry matter in the dough composition.22. The dough composition of claim 18, wherein the dough compositionfurther comprises one or more additional ingredients selected from thegroup consisting of protein, eggs, oxidants, sugars, fat and salts. 23.The dough composition of claim 18, wherein the dough composition furthercomprises an emulsifier.
 24. The dough composition of claim 18, whereinthe dough composition further comprises a leavening agent.
 25. The doughcomposition of claim 18, wherein the dough composition further comprisesa maltogenic amylase.